Nowadays, the model of loop domain organization of eukaryotic chromosomes is well accepted (Boulikas T, “Nature of DNA sequences at the attachment regions of genes to the nuclear matrix”, J. Cell Biochem., 52:14-22, 1993). According to this model chromatin is organized in loops that span 50-100 kb attached to the nuclear matrix, a proteinaceous network made up of RNPs and other nonhistone proteins (Bode J, Stengert-Iber M, Kay V, Schalke T and Dietz-Pfeilstetter A, Crit. Rev. Euk. Gene Exp., 6:115-138, 1996).
The DNA regions attached to the nuclear matrix are termed SAR or MAR for respectively scaffold (during metaphase) or matrix (interphase) attachment regions (Hart C and Laemmli U (1998), “Facilitation of chromatin dynamics by SARs” Curr Opin Genet Dev 8, 519-525.)
As such, these regions may define boundaries of independent chromatin domains, such that only the encompassing cis-regulatory elements control the expression of the genes within the domain.
However, their ability to fully shield a chromosomal locus from nearby chromatin elements, and thus confer position-independent gene expression, has not been seen in stably transfected cells (Poljak L, Seum C, Mattioni T and Laemmli U. (1994) “SARs stimulate but do not confer position independent gene expression”, Nucleic Acids Res 22, 4386-4394). On the other hand, MAR (or S/MAR) sequences have been shown to interact with enhancers to increase local chromatin accessibility (Jenuwein T, Forrester W, Fernandez-Herrero L, Laible G, Dull M, and Grosschedl R. (1997) “Extension of chromatin accessibility by nuclear matrix attachment regions” Nature 385, 269-272). Specifically, MAR elements can enhance expression of heterologous genes in cell culture lines (Kalos M and Fournier R (1995) “Position-independent transgene expression mediated by boundary elements from the apolipoprotein B chromatin domain” Mol Cell Biol 15, 198-207), transgenic mice (Castilla J, Pintado B, Sola, I, Sanchez-Morgado J, and Enjuanes L (1998) “Engineering passive immunity in transgenic mice secreting virus-neutralizing antibodies in milk” Nat Biotechnol 16, 349-354) and plants (Allen G, Hall G J, Michalowski S, Newman W, Spiker S, Weissinger A, and Thompson W (1996), “High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco” Plant Cell 8, 899-913). The utility of MAR sequences for developing improved vectors for gene therapy is also recognized (Agarwal M, Austin T, Morel F, Chen J, Bohnlein E, and Plavec I (1998), “Scaffold attachment region-mediated enhancement of retroviral vector expression in primary T cells” J Virol 72, 3720-3728).
Recently, it has been shown that chromatin-structure modifying sequences including MARs, as exemplified by the chicken lysozyme 5′ MAR is able to significantly enhance reporter expression in pools of stable Chinese Hamster Ovary (CHO) cells (Zahn-Zabal M, et al., “Development of stable cell lines for production or regulated expression using matrix attachment regions” J Biotechnol, 2001, 87(1): p. 29-42). This property was used to increase the proportion of high-producing clones, thus reducing the number of clones that need to be screened. These benefits have been observed both for constructs with MARs flanking the transgene expression cassette, as well as when constructs are co-transfected with the MAR on a separate plasmid. However, expression levels upon co-transfection with MARs were not as high as those observed for a construct in which two MARs delimit the transgene expression unit. A third and preferable process was shown to be the transfection of transgenes with MARs both linked to the transgene and on a separate plasmid (Girod et al., submitted for publication). However, one persisting limitation of this technique is the quantity of DNA that can be transfected per cell. Many multiples transfection protocols have been developed in order to achieve a high transfection efficiency to characterize the function of genes of interest. The protocol applied by Yamamoto et al, 1999 (“High efficiency gene transfer by multiple transfection protocol”, Histochem. J. 31(4), 241-243) leads to a transfection efficiency of about 80% after 5 transfections events, whereas the conventional transfection protocol only achieved a rate of <40%. While this technique may be useful when one wishes to increase the proportion of expressing cells, it does not lead to cells with a higher intrinsic productivity. Therefore, it cannot be used to generate high producer monoclonal cell lines. Hence, the previously described technique has two major drawbacks:                i) this technique does not generate a homogenous population of transfected cells, since it cannot favor the integration of further gene copy, nor does it direct the transgenes to favorable chromosomal loci,        ii) the use of the same selectable marker in multiple transfection events does not permit the selection of doubly or triply transfected cells.        
In patent application WO02/074969, the utility of MARs for the development of stable eukaryotic cell lines has also been demonstrated. However, this application does not disclose neither any conserved homology for MAR DNA element nor any technique for predicting the ability for a DNA sequence to be a MAR sequence.
In fact no clear-cut MAR consensus sequence has been found (Boulikas T, “Nature of DNA sequences at the attachment regions of genes to the nuclear matrix”, J. Cell Biochem., 52:14-22, 1993) but evolutionarily, the structure of these sequences seem to be functionally conserved in eukaryotic genomes, since animal MARs can bind to plant nuclear scaffolds and vice versa (Mielke C, Kohwi Y, Kohwi-Shigematsu T and Bode J, “Hierarchical binding of DNA fragments derived from scaffold-attached regions: correlation of properties in vitro and function in vivo”, Biochemistry, 29:7475-7485, 1990).
The identification of MARs by biochemical studies is a long and unpredictable process; various results can be obtained depending on the assay (Razin S V, “Functional architecture of chromosomal DNA domains”, Crit Rev Eukaryot Gene Expr., 6:247-269, 1996). Considering the huge number of expected MARs in a eukaryotic genome and the amount of sequences issued from genome projects, a tool able to filter potential MARS in order to perform targeted experiments would be greatly useful.
Currently two different predictive tools for MARs are available via the Internet. The first one, MAR-Finder (Singh G B, Kramer J A and Krawetz S A, “Mathematical model to predict regions of chromatin attachment to the nuclear matrix”, Nucleic Acid Research, 25:1419-1425, 1997) is based on set of patterns identified within several MARs and a statistical analysis of the co-occurrence of these patterns. MAR-Finder predictions are dependent of the sequence context, meaning that predicted MARs depend on the context of the submitted sequence. The other predictive software, SMARTest (Frisch M, Frech K, Klingenhoff A, Cartharius K, Liebich I and Werner T, “In silico prediction of scaffold/matrix attachment regions in large genomic sequences”, Genome Research, 12:349-354, 2001), use weight-matrices derived from experimentally identified MARs. SMARTest is said to be suitable to perform large-scale analyses. But actually aside its relative poor specificity, the amount of hypothetical MARs rapidly gets huge when doing large scale analyses with it, and in having no way to increase its specificity to restrain the number of hypothetical MARs, SMARTest becomes almost useless to screen for potent MARs form large DNA sequences.
Some other softwares, not available via the Internet, also exists; they are based as well on the frequency of MAR motifs (MRS criterion; Van Drunen C M et al., “A bipartite sequence element associated with matrix/scaffold attachment regions”, Nucleic Acids Res, 27:2924-2930, 1999), (ChrClass; Glazko G V et al., “Comparative study and prediction of DNA fragments associated with various elements of the nuclear matrix”, Biochim. Biophys. Acta, 1517:351-356, 2001) or based on the identification of sites of stress-induced DNA duplex (SIDD; Benham C and al., “Stress-induced duplex DNA destabilization in scaffold/matrix attachment regions”, J. Mol. Biol., 274:181-196, 1997). However, their suitability to analyze complete genome sequences remains unknown, and whether these tools may allow the identification of protein production-increasing sequences has not been reported.
Furthermore, due to the relatively poor specificity of these softwares (Frisch M, Frech K, Klingenhoff A, Cartharius K, Liebich I and Werner T, “In silico prediction of scaffold/matrix attachment regions in large genomic sequences”, Genome Research, 12:349-354, 2001), the amount of hypothetical MARs identified in genomes rapidly gets unmanageable when doing large scale analyses, especially if most of these have no or poor activity in practice. Thus, having no way to increase prediction specificity to restrain the number of hypothetical MARs, many of the available programs become almost useless to identify potent genetic elements in view of efficiently increasing recombinant protein production.
Since all the above available predictive methods have some drawbacks that prevent large-scale analyses of genomes to identify reliably novel and potent MARs, the object of this invention is to 1) understand the functional features of MARs that allow improved recombinant protein expression; 2) get a new Bioinformatic tool compiling MAR structural features as a prediction of function, in order to 3) perform large scale analyses of genomes to identify novel and more potent MARs, and, finally 4) to demonstrate improved efficiency to increase the production of recombinant proteins from eukaryotic cells or organisms when using the newly identified MAR sequences.